1-1. Recombinant Vector

Mutation was induced in acetylglutamate kinase to enhance the biosynthesis pathway in the strain. In the method used in this Example, the argB gene encoding acetylglutamate kinase was subjected to site-directed mutagenesis in order to increase expression of the gene. Glutamic acid at amino acid position 258 of the acetylglutamate kinase encoded by the argB gene was substituted with leucine, and the left arm portion (1,614 bp) and the right arm portion (854 bp) with respect to the center of the argB gene on the Corynebacterium glutamicum genome were amplified by PCR and ligated together by overlap PCR, followed by cloning into a pk19msb vector. The resulting plasmid was named pk19msb+argB-E258L (see FIG. 1). To construct the plasmid, the primers shown in Table 1 below were used for amplification of each gene fragment.

TABLE 1

Primer (5′→3′)
SEQ ID NO

Amplification
argB-F1
tgattacgcc actgacacggtggataaggaaac
9

primers for left
argB-F2
actgacacggtggataaggaaac
10

homologous arm of
E258L-R1
agcagcaacaccgagtgc
11

argB
E258L-R2
ggtcaaaagcagcagcaaca
12

Amplification
E258L-F1
gcttttgacc atgggtggaattggcacg
13

primers for right
E258L-F2
atgggtggaattggcacg
14

homologous arm of
argB-R1
catcgggaacaacgccat
15

argB
argB-R2
atggtcaccacatcgggaa
16

Using the above-described primers, PCR was performed under the following conditions. PCR was performed using a Thermocycler (TP600, TAKARA BIO Inc., Japan) in a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), 1 μM oligonucleotide, 10 ng of the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, and 1 unit of pfu-X DNA polymerase mixture (Solgent). PCR was performed for 25 to 30 cycles, each consisting of (i) denaturation at 94° C. for 30 sec, (ii) annealing at 58° C. for 30 sec, and (iii) extension at 72° C. for 1 to 2 min (a polymerization time of 2 min per kb).

The gene fragments prepared as described above were cloned into a pk19msb vector by self-assembly cloning. The vector was transformed into E. coli DH5a which was then plated on an LB-agar plate containing 50 μg/ml of kanamycin, and cultured at 37° C. for 24 hours. The finally formed colony was isolated and whether the insert was exactly present in the vector was checked. Next, the vector was isolated and used for recombination of the Corynebacterium glutamicum strain.

As a process commonly performed in the above method, the corresponding genes were amplified by PCR from the genomic DNA of Corynebacterium glutamicum ATCC 13032 and inserted into the pk19msb vector by the self-assembled cloning method according to the strategy, and the resulting plasmid was selected in E coli DH5a. For chromosomal base substitution, the gene fragments were amplified individually and ligated together by overlap PCR, thereby preparing the desired DNA fragment. For genetic manipulation, Ex Taq polymerase (Takara) and Pfu polymerase (Solgent) were used as PCR polymerases, and various restriction enzymes and DNA modifying enzymes purchased from NEB were used, and they were used according to manufacturer's provided buffers and protocols.

1-2. Corynebacterium glutamicum Mutant Strain AB1

Mutant strain AB1 was constructed using a cloning vector. The cloning vector was prepared at a final concentration of 1 μg/μl or more and electroporated into a Corynebacterium glutamicum 14GR strain (see Tauch et al., FEMS Microbiology letters 123 (1994) 343-347) to induce first recombination. In this case, the electroporated strain was plated on an agar medium containing 50 μg/μl of kanamycin, colonies were isolated, and then whether the vector was properly inserted at the desired position on the genome was checked by PCR and sequencing. Each of the isolated strains was inoculated again in a liquid medium to induce second recombination, cultured overnight or more, and then plated on an agar medium containing 10% sucrose, and colonies were isolated. Whether the finally isolated colonies were resistant to kanamycin was checked, and then whether mutation was introduced into the acetylglutamate kinase in the strains having no antibiotic resistance was checked by sequencing (see Schafer et al., Gene 145 (1994) 69-73). As a result, Corynebacterium glutamicum mutant strain AB1 capable of producing L-arginine was constructed, which has a substitution of leucine for glutamic acid at position 258 in the amino acid sequence of acetylglutamate kinase (SEQ ID NO: 2).

Example 2. Construction of Corynebacterium glutamicum Mutant Strain

A Corynebacterium glutamicum mutant strain having enhanced activity of acetylglutamate kinase was constructed in the same manner as in Example 1, a recombinant vector (pk19msb+argB-E258A) containing an argB gene encoding an acetylglutamate kinase having a substitution of alanine for glutamic acid at amino acid position 258 was used.

More specifically, glutamic acid at amino acid position 258 of the acetylglutamate kinase encoded by the argB gene was substituted with alanine, and the left arm portion (1,614 bp) and the right arm portion (854 bp) with respect to the center of the argB gene on the Corynebacterium glutamicum genome were amplified by PCR and ligated together by overlap PCR, followed by cloning into a pk19msb vector. The resulting plasmid was named pk19msb+argB-E258A (see FIG. 2). To construct the plasmid, the primers shown in Table 2 below were used for amplification of each gene fragment.

TABLE 2

Primer (5′→3′)
SEQ ID NO

Amplification
argB-F1
tgattacgcc actgacacggtggataaggaaac
9

primers for left
argB-F2
actgacacggtggataaggaaac
10

homologous arm of
E258A-R1
gccagcaacaccgagt
17

argB
E258A-R2
ggtcaaaagcgccagcaaca
18

Amplification
E258L-F1
gcttttgacc atgggtggaattggcacg
13

primers for right
E258L-F2
atgggtggaattggcacg
14

homologous arm of
argB-R1
catcgggaacaacgccat
15

argB
argB-R2
atggtcaccacatcgggaa
16

As a result, Corynebacterium glutamicum mutant strain AB2 capable of producing L-arginine was constructed, which has a substitution of alanine for glutamic acid at position 258 in the amino acid sequence of acetylglutamate kinase (SEQ ID NO: 2).

Example 3. Construction of Corynebacterium glutamicum Mutant Strain

A Corynebacterium glutamicum mutant strain having enhanced activity of acetylglutamate kinase was constructed in the same manner as in Example 1, a recombinant vector (pk19msb+argB-H245D) containing an argB gene encoding an acetylglutamate kinase having a substitution of aspartic acid for histidine at amino acid position 245 was used.

More specifically, histidine at amino acid position 245 of the acetylglutamate kinase encoded by the argB gene was substituted with aspartic acid, and the left arm portion (1,583 bp) and the right arm portion (885 bp) with respect to the center of the argB gene on the Corynebacterium glutamicum genome were amplified by PCR and ligated together by overlap PCR, followed by cloning into the recombinant vector pk19msb. The resulting plasmid was named pk19msb+argB-H245D (see FIG. 3). To construct the plasmid, the primers shown in Table 3 below were used for amplification of each gene fragment.

TABLE 3

Primer (5′→3′)
SEQ ID NO

Amplification
argB-F1
tgattacgcc actgacacggtggataaggaaac
9

primers for left
argB-F2
actgacacggtggataaggaaac
10

homologous arm of
H245D-R1
caatgacatcagcagcgctta
19

argB
H245D-R2
atgcggccgtcaatgac
20

Amplification
H245D-F1
acggccgcat cgcgcactcggtgttgct
21

primers for right
H245D-F2
cgcgcactcggtgttgct
22

homologous arm of
argB-R1
catcgggaacaacgccat
15

argB
argB-R2
atggtcaccacatcgggaa
16

As a result, Corynebacterium glutamicum mutant strain AB3 capable of producing L-arginine was constructed, which has a substitution of aspartic acid for histidine at position 245 in the amino acid sequence of acetylglutamate kinase (SEQ ID NO: 2).

Experimental Example 1. Evaluation of L-Arginine Productivities of Corynebacterium glutamicum Mutant Strains

The L-arginine productivities of the Corynebacterium glutamicum mutant strains AB1 to AB3 constructed in Examples 1 to 3 were evaluated in comparison with that of the parent strain.

Each strain was patched on a flask solid seed medium and cultured at 30° C. for 24 hours. Each cultured colony was inoculated into a 10-ml flask titer medium and cultured at 200 rpm at 32° C. for 30 hours. The compositions of the media used here are shown in Table 4 below. After completion of the culturing, each culture was diluted 100-fold with distilled water and filtered through a 0.45-μm filter, and then the amount of L-arginine produced was analyzed using high-performance liquid chromatography (HPLC) (Agilent Technologies 1260 Infinity, Agilent Technologies) equipped with a column (DionexIonPac™ CS12A) and a UV detector (195 mm), and the results are shown in Table 5 below. In Table 5, L-arginine (%) denotes the amount (percentage) of arginine produced by each strain, and fermentation yield (Yp/s) (%) denotes the amount of L-arginine produced per glucose consumed.

TABLE 4

Flask
10.5 g of 98% glucose, 1 g of beef extract, 4 g of

solid seed
yeast extract, 2 g of polypeptone, 2 g of NaCl, 40 g

medium
of (NH₄)₂SO₄and 20 g of agar

(per L)

Flask titer
120 g of 98% glucose, 1 g of MgSO₄, 2 g of KH₂PO₄,

medium
45 g of (NH₄)₂SO₄, 20 mg of FeSO₄, 20 mg of MnSO₄,

(per L)
100 μg of biotin, 100 μg of thiamine,

4 g of YSP and 2 g of urea